Bjørg Haldorsen

Genetic methods for detection of antimicrobial resistance

Accurate and rapid diagnostic methods are needed to guide antimicrobial therapy and infection control interventions. Advances in real‐time PCR have provided a user‐friendly, rapid and reproducible testing platform catalysing an increased use of genetic assays as part of a wider strategy to minimize the development and spread of antimicrobial‐resistant bacteria. In this review we outline the principal features of genetic assays in the detection of antimicrobial resistance, their advantages and limitations, and discuss specific applications in the detection of methicillin‐resistant Staphylococcus aureus, glycopeptide‐resistant enterococci, aminoglycoside resistance in staphylococci and enterococci, broad‐spectrum resistance to β‐lactam antibiotics in gram‐negative bacteria, as well as genetic elements involved in the assembly and spread of antimicrobial resistance.

Effects of Phenotype and Genotype on Methods for Detection of Extended-Spectrum-β-Lactamase-Producing Clinical Isolates of Escherichia coli and Klebsiella pneumoniae in Norway

ABSTRACT Consecutive clinical isolates of Escherichia coli (n = 87) and Klebsiella pneumoniae (n = 25) with reduced susceptibilities to oxyimino-cephalosporins (MICs > 1 mg/liter) from 18 Norwegian laboratories during March through October 2003 were examined for blaTEM/SHV/CTX-M extended-spectrum-β-lactamase (ESBL) genes, oxyimino-cephalosporin MIC profiles, ESBL phenotypes (determined by the ESBL Etest and the combined disk and double-disk synergy [DDS] methods), and susceptibility to non-β-lactam antibiotics. Multidrug-resistant CTX-M-15-like (n = 23) and CTX-M-9-like (n = 15) ESBLs dominated among the 50 ESBL-positive E. coli isolates. SHV-5-like (n = 9) and SHV-2-like (n = 4) ESBLs were the most prevalent in 19 ESBL-positive K. pneumoniae isolates. Discrepant ESBL phenotype test results were observed for one major (CTX-M-9) and several minor (TEM-128 and SHV-2/-28) ESBL groups and in SHV-1/-11-hyperproducing isolates. Negative or borderline ESBL results were observed when low-MIC oxyimino-cephalosporin substrates were used to detect clavulanic acid (CLA) synergy. CLA synergy was detected by the ESBL Etest and the DDS method but not by the combined disk method in SHV-1/-11-hyperproducing strains. The DDS method revealed unexplained CLA synergy in combination with aztreonam and cefpirome in three E. coli strains. The relatively high proportion of ESBL-producing E. coli organisms with a low ceftazidime MIC in Norway emphasizes that cefpodoxime alone or both cefotaxime and ceftazidime should be used as substrates for ESBL detection.

Molecular characterization of CTX-M-15-producing clinical isolates of Escherichia coli reveals the spread of multidrug-resistant ST131 (O25:H4) and ST964 (O102:H6) strains in Norway.

Nationwide, CTX‐M‐producing clinical Escherichia coli isolates from the Norwegian ESBL study in 2003 (n=45) were characterized on strain and plasmid levels. BlaCTX‐M allele typing, characterization of the genetic environment, phylogenetic groups, pulsed field gel electrophoresis (PFGE), serotyping and multilocus sequence typing were performed. Plasmid analysis included S1‐nuclease‐PFGE, polymerase chain reaction‐based replicon typing, plasmid transfer and multidrug resistance profiling. BlaCTX‐M‐15 (n=23; 51%) and blaCTX‐M‐14 (n=11; 24%) were the major alleles of which 18 (78%) and 6 (55%), respectively, were linked to ISEcp1. Thirty‐two isolates were of phylogenetic groups B2 and D. Isolates were of 29 different XbaI‐PFGE‐types including six regional clusters. Twenty‐three different O:H serotypes were found, dominated by O25:H4 (n=9, 20%) and O102:H6 (n=9, 20%). Nineteen different STs were identified, where ST131 (n=9, 20%) and ST964 (n=7, 16%) were dominant. BlaCTX‐M was found on ≥100 kb plasmids (39/45) of 10 different replicons dominated by IncFII (n=39, 87%), FIB (n=20, 44%) and FIA (n=19, 42%). Thirty‐nine isolates (87%) displayed co‐resistance to other classes of antibiotics. A transferable CTX‐M phenotype was observed in 9/14 isolates. This study reveals that the majority of CTX‐M‐15‐expressing strains in Norway are part of the global spread of multidrug‐resistant ST131 and ST‐complex 405, associated with ISEcp1 on transferrable IncFII plasmids.

Species identification and molecular characterization of Acinetobacter spp. blood culture isolates from Norway

OBJECTIVES The study investigated the species distribution, antibiotic susceptibility patterns and genotypic resistance characteristics of 113 consecutive blood culture isolates of Acinetobacter species collected between 2005 and 2007 throughout Norway. METHODS Species identification was performed by partial rpoB sequence analysis, and verified by 16S rDNA and recA sequence analyses. Susceptibility testing was performed by agar disc diffusion and Etest. Distribution of OXA carbapenemase genes and epidemic clonality of Acinetobacter baumannii isolates were detected by PCR assays. Analyses of blaOXA-51-like variants and quinolone resistance-determining regions (QRDRs) were done by sequencing. RESULTS The most prevalent species in the collection were Acinetobacter genomic species (gen. sp.) 13TU (46.9%) and Acinetobacter gen. sp. 3 (19.5%), followed by A. baumannii (8.8%) and Acinetobacter lwoffii/Acinetobacter gen. sp. 9 (7.1%). Carbapenem resistance was observed in one blaOXA-23-like-positive A. baumannii isolate. Quinolone resistance was detected in five isolates from the Acinetobacter calcoaceticus-A. baumannii complex, of which two had point mutations in the QRDRs, including one novel ParC mutation. None of the A. baumannii isolates belonged to European/international clones I, II or III. Six blaOXA-51-like variants, including two novel variants, were identified. CONCLUSIONS Acinetobacter gen. sp. 13TU and Acinetobacter gen. sp. 3 were predominant in Norwegian blood cultures, in contrast to in other countries where A. baumannii has dominated. The study demonstrated the importance of genotypic identification to determine the exact epidemiology of non-baumannii Acinetobacter species.

Sporadic occurrence of CMY-2-producing multidrug-resistant Escherichia coli of ST-complexes 38 and 448, and ST131 in Norway

Clinical isolates of Escherichia coli with reduced susceptibility to oxyimino-cephalosporins and not susceptible to clavulanic acid synergy (n = 402), collected from Norwegian diagnostic laboratories in 2003-2007, were examined for the presence of plasmid-mediated AmpC beta-lactamases (PABLs). Antimicrobial susceptibility testing was performed for beta-lactam and non-beta-lactam antibiotics using Etest and Vitek2, respectively. The AmpC phenotype was confirmed using the boronic acid test. PABL-producing isolates were detected using ampC multiplex-PCR and examined by bla(AmpC) sequencing, characterization of the bla(AmpC) genetic environment, phylogenetic grouping, XbaI- pulsed-field gel electrophoresis (PFGE), multi-locus sequence typed (MLST), plasmid profiling and PCR-based replicon typing. For the PABL-positive isolates (n = 38), carrying bla(CMY-2) (n = 35), bla(CMY-7) (n = 1) and bla(DHA-1) (n = 2), from out- (n = 23) and in-patients (n = 15), moderate-high MICs of beta-lactams, except cefepime and carbapenems, were determined. All isolates were resistant to trimethoprim-sulphamethoxazole. Multidrug resistance was detected in 58% of the isolates. The genes bla(CMY-2) and bla(CMY-7) were linked to ISEcp1 upstream in 32 cases and in one case, respectively, and bla(DHA-1) was linked to qacEDelta1sul1 upstream and downstream in one case. Twenty isolates were of phylogenetic groups B2 or D. Thirty-three XbaI-PFGE types, including three clusters, were observed. Twenty-five sequence types (ST) were identified, of which ST complexes (STC) 38 (n = 7), STC 448 (n = 5) and ST131 (n = 4) were dominant. Plasmid profiling revealed 1-4 plasmids (50-250 kb) per isolate and 11 different replicons in 37/38 isolates; bla(CMY-2) was carried on transferable multiple-replicon plasmids, predominantly of Inc groups I1 (n = 12), FII (n = 10) and A/C (n = 7). Chromosomal integration was observed for bla(CMY-2) in ten strains. CMY-2 is the dominant PABL type in Norway and is associated with ISEcp1 and transferable, multiple-replicon IncI1, IncA/C, or IncFII plasmids in nationwide strains of STC 448, STC 38 and ST131.

Nosocomial outbreak of CTX-M-15-producing E. coli in Norway.

Seven E. coli isolates expressing resistance to 3rd generation cephalosporins were recovered from blood (n=2), kidney and lung tissue (n=1), and urinary tract (n=4) samples from seven patients hospitalised or recently discharged from the Divisions of Geriatrics and Pulmonary Medicine, Central Hospital of Rogaland, between July and September 2004. All isolates expressed a typical ESBL‐cefotaximase profile (cefotaxime MIC>ceftazidime MIC) with clavulanic acid synergy. A blaCTX‐M‐15 genotype was confirmed in six strains that were coresistant to gentamicin, nitrofurantoin, trimethoprim‐sulfamethoxazole and ciprofloxacin. A blaCTX‐M‐3 genotype was detected in the last strain. XbaI‐PFGE patterns of the six blaCTX‐M‐15 isolates revealed a clonal relationship. BlaCTX‐M‐15 strains were also positive for the ISEcp1‐like insertion sequences that have been shown to be involved in the mobilization of blaCTX‐M. Further analyses revealed two blaCTX‐M‐15‐positive E. coli urinary isolates clonally related to the outbreak strain from two different patients at the same divisions in January and February 2004. These patients were later re‐hospitalised and one had E. coli with an ESBL‐cefotaximase profile in sputum and nasopharyngeal specimen during the outbreak period. Clinical evaluation suggests that the CTX‐M‐producing E. coli strains contributed to death in three patients due to delayed efficient antimicrobial therapy. The outbreak emphasises the epidemic potential of multiple‐antibiotic‐resistant CTX‐M‐15‐producing E. coli also in a country with low antibiotic usage and low prevalence of antimicrobial resistance.

Comparison of disk diffusion, Etest and VITEK2 for detection of carbapenemase‐producing Klebsiella pneumoniae with the EUCAST and CLSI breakpoint systems

The aim of this study was to compare CLSI and EUCAST MIC and disk diffusion carbapenem breakpoints for the detection of carbapenemase-producing Klebsiella pneumoniae. K. pneumoniae strains with known KPC (n = 31) or VIM (n = 20) carbapenemases were characterized by disk diffusion (Oxoid) and Etest (bioMérieux) vs. imipenem, meropenem and ertapenem, and with VITEK2 (bioMérieux, five different cards). Extended-spectrum β-lactamase (ESBL) testing was performed with VITEK2 (bioMérieux), ESBL combination disks (Becton Dickinson) and the ESBL Etest (bioMérieux). With CLSI and EUCAST MIC breakpoints, respectively, 11 and seven of the strains were susceptible to imipenem, 12 and eight to meropenem, and seven and none to ertapenem. The EUCAST epidemiological cut-off (ECOFF) values for meropenem and ertapenem identified all carbapenemase producers, whereas the imipenem ECOFF failed in five strains. All carbapenemase producers were detected with EUCAST disk diffusion breakpoints for ertapenem and meropenem, and four strains were susceptible to imipenem. CLSI disk diffusion breakpoints characterized 18 (imipenem), 14 (meropenem) and three (ertapenem) isolates as susceptible. When cards with a single carbapenem were used, detection failures with VITEK2 were four for imipenem, none for meropenem and one for ertapenem. Cards containing all three carbapenems had one to two failures. With ESBL combination disks, 21/31 KPC producers and 2/20 VIM producers were positive. With VITEK2, no VIM producers and between none and seven KPC producers were ESBL-positive. All carbapenemase producers were detected with the meropenem MIC ECOFF, or the clinical EUCAST breakpoint for ertapenem. EUCAST disk diffusion breakpoints for meropenem and ertapenem detected all carbapenemase producers. VITEK2 had between none and four failures in detecting carbapenemase producers, depending on the antibiotic card.

The AmpC phenotype in Norwegian clinical isolates of Escherichia coli is associated with an acquired ISEcp1-like ampC element or hyperproduction of the endogenous AmpC

OBJECTIVES The aim of the study was to examine resistance mechanisms associated with an AmpC phenotype in Norwegian clinical isolates of Escherichia coli. METHODS Clinical E. coli isolates (n = 106) with reduced susceptibility to third-generation cephalosporins without clavulanic acid synergy were collected from 12 Norwegian laboratories from 2003 to 2005. Twenty-two isolates with an AmpC phenotype were selected for further characterization by PFGE, isoelectric focusing, different PCR-based techniques, DNA sequencing, AmpC qRT-PCR, transfer studies and plasmid analyses. RESULTS The 22 isolates were not clonally related by the PFGE analysis. All isolates expressed a beta-lactamase with a pI of 9.0-9.2. Ten isolates contained a bla(CMY) gene, which was linked to an ISEcp1-like element in all cases. Twelve isolates had mutations or insertions in the promoter or the attenuator regions, leading to increased expression of the chromosomal ampC gene. One of these isolates had an ISEc10 element inserted upstream of the chromosomal ampC gene. CONCLUSIONS This is the first molecular study of Norwegian clinical E. coli isolates with an AmpC phenotype. Resistance was mediated either by expression of bla(CMY) from acquired ISEcp1-like-bla(CMY) elements, or by mutations or insertions in the chromosomal ampC gene control region leading to hyperproduction of the endogenous AmpC enzyme. There was no correlation between the level of ampC mRNA and the MICs of cephalosporins.

Clinical isolates of Staphylococcus aureus from the Arkhangelsk region, Russia: antimicrobial susceptibility, molecular epidemiology, and distribution of Panton‐Valentine leucocidin genes

A total of 91 consecutive clinical isolates of Staphylococcus aureus were collected at the Regional Hospital of Arkhangelsk, Russia, from May to December 2004, and examined for antimicrobial susceptibility, methicillin resistance and presence of Panton‐Valentine leucocidin (PVL) genes. Epidemiological typing was performed by pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Methicillin‐resistant S. aureus (MRSA) isolates were examined by staphylococcal cassette chromosome mec (SCCmec) typing. High‐to‐moderate rates of resistance to penicillin (β‐lactamase production; 93%), tetracycline (40%), erythromycin and clindamycin (32%) were observed. Forty out of ninety‐one (44%) isolates were positive for PVL genes. Thirty‐six (40%) PVL‐positive methicillin‐susceptible S. aureus (MSSA) strains were shown by PFGE and MLST typing (ST121, ST681, ST837) to be part of a nosocomial outbreak caused by clonal complex (CC) 121. PFGE, MLST and SCCmec typing revealed three MRSA clones. Sequence type (ST) 239‐III (n=11), ST1097‐III (n=1) and ST8‐IV (n=3) belong to CC8 of epidemic multiresistant MRSA, whereas ST426‐MRSA‐IV/CC395 (n=1) has not been reported previously. All MRSA strains were PVL negative. The overall results underline the necessity of microbiological sampling, antimicrobial susceptibility testing, and epidemiological typing as a rational basis for antimicrobial treatment of S. aureus infections, and infection control measures to limit the spread of multiresistant MRSA and epidemic MSSA clones.

Performance of the EUCAST disk diffusion method, the CLSI agar screen method, and the Vitek 2 automated antimicrobial susceptibility testing system for detection of clinical isolates of enterococci with low- and medium-level VanB-type vancomycin resistance: A multicenter study

ABSTRACT Different antimicrobial susceptibility testing methods to detect low-level vancomycin resistance in enterococci were evaluated in a Scandinavian multicenter study (n = 28). A phenotypically and genotypically well-characterized diverse collection of Enterococcus faecalis (n = 12) and Enterococcus faecium (n = 18) strains with and without nonsusceptibility to vancomycin was examined blindly in Danish (n = 5), Norwegian (n = 13), and Swedish (n = 10) laboratories using the EUCAST disk diffusion method (n = 28) and the CLSI agar screen (n = 18) or the Vitek 2 system (bioMérieux) (n = 5). The EUCAST disk diffusion method (very major error [VME] rate, 7.0%; sensitivity, 0.93; major error [ME] rate, 2.4%; specificity, 0.98) and CLSI agar screen (VME rate, 6.6%; sensitivity, 0.93; ME rate, 5.6%; specificity, 0.94) performed significantly better (P = 0.02) than the Vitek 2 system (VME rate, 13%; sensitivity, 0.87; ME rate, 0%; specificity, 1). The performance of the EUCAST disk diffusion method was challenged by differences in vancomycin inhibition zone sizes as well as the experience of the personnel in interpreting fuzzy zone edges as an indication of vancomycin resistance. Laboratories using Oxoid agar (P < 0.0001) or Merck Mueller-Hinton (MH) agar (P = 0.027) for the disk diffusion assay performed significantly better than did laboratories using BBL MH II medium. Laboratories using Difco brain heart infusion (BHI) agar for the CLSI agar screen performed significantly better (P = 0.017) than did those using Oxoid BHI agar. In conclusion, both the EUCAST disk diffusion and CLSI agar screening methods performed acceptably (sensitivity, 0.93; specificity, 0.94 to 0.98) in the detection of VanB-type vancomycin-resistant enterococci with low-level resistance. Importantly, use of the CLSI agar screen requires careful monitoring of the vancomycin concentration in the plates. Moreover, disk diffusion methodology requires that personnel be trained in interpreting zone edges.